Project Summary/Abstract Cis-regulatory long noncoding RNAs (lncRNAs) have the unique capability to regulate the expression of neighboring genes. Historically studied in the context of dosage compensation, there is mounting evidence that cis-regulatory lncRNAs may also play widespread roles in normal physiology and disease development. However, their functions and mechanisms of action have remained poorly characterized. The study proposed herein aims to understand the role of cis-regulation in cancer biology by focusing on two lncRNAs, lncRNA- Gadd45g and an alternative isoform of Pvt1, Pvt1alt. In preliminary studies, we have established that these lncRNAs are directly regulated by the central tumor suppressor factor p53, that they act in cis to regulate their neighboring genes, namely the tumor suppressor Gadd45g and the oncogene c-Myc, and that Pvt1alt is involved in suppressing clonal growth. Based on these exciting data, we have hypothesized that cis-regulatory lncRNAs provide an unappreciated layer of regulation in the p53 network and may mediate p53-dependent stress responses and tumor suppressive functions. To address this hypothesis, in Aim 1, we propose to perform a comprehensive series of experiments at the cellular level, which will test whether genetic inhibition of lncRNA-Gadd45g and Pvt1alt in primary and transformed cell lines affects p53-dependent transcriptional and cellular responses to stress. In parallel, we will examine whether genetic inhibition of the two lncRNAs in developing tumors in a mouse model of lung cancer promotes tumor development, as shown for p53. Together, this set of experiments will establish the physiological relevance of cis-regulatory lncRNAs in the context of the p53 network in vitro and in vivo. In Aims 2 and 3, we will elucidate the mechanism of action of lncRNA- Gadd45g and Pvt1alt. Aim 2 applies an innovative genetic approach for transcript-specific degradation, developed by us, in order to determine the functional element of cis-regulation by dissociating the accumulation of the lncRNA transcript from the act of its transcription and from the underlying DNA elements in the locus. Findings from this set of studies will advance the field by clarifying a controversy about the central player in local gene expression control and may elucidate how cis-regulatory relationships become disrupted in disease states, including cancer. Finally, in an effort to define universal principles as well as locus-specific features of cis regulation, in Aim 3, we will determine whether cis-acting lncRNAs impact local transcriptional and epigenetic events by examining the kinetics of transcription, the epigenetic state, and the chromatin architecture in their respective loci. In summary, the studies proposed here take advantage of rigorous genetic approaches, elegant functional assays, and state-of-the art molecular tools to address the contribution of cis- regulatory lncRNAs to the p53 tumor suppressor pathway. The broad significance of this work lies in its potential to expand our understanding of the transcriptional, epigenetic, and chromatin re-organization events that take place during the physiological response to stress and become frequently perturbed in cancer.